Group A Streptococcus (GAS, S. pyogenes) remains a major public health threat. This widespread Gram- positive bacterial pathogen causes acute invasive diseases and gives rise to severe autoimmune sequelae, and is responsible for morbidity and mortality on a global scale. An essential virulence factor of GAS, the antigenically variable M protein, enables the bacterium to evade opsonophagocytic killing by the immune system. The M protein confers this indispensable function of phagocyte resistance by recruiting specific soluble human proteins to the GAS surface that block the deposition of the major opsonin C3b as well as antigen- specific opsonic antibodies ? namely, C4b-binding protein (C4BP), factor H (FH), and fibrinogen (Fg). In some GAS strains, an M-like protein serves this function. More than 220 M types are known, and while most M protein types bind C4BP, FH, or Fg, or a combination of these, no consensus binding motif is evident in M and M-like proteins for any of these human proteins, except most recently for C4BP due to our breakthrough. This is because the exposed portion of the M protein that recruits these anti-opsonic human proteins is sequence variable. While inhibition of these interactions has been shown to render GAS sensitive to immune killing, the lack of consensus binding motifs has hindered the therapeutic goal of targeting these interactions. Our recent breakthrough, reported in Buffalo et al., offers a solution to this problem. In effect, we found that hidden within the variable region of many M proteins is a three-dimensional (3D) pattern that is conserved for binding C4BP. This 3D C4BP-binding pattern in the M protein is dispersed (or hidden) within a sea of variable amino acids, which makes the 3D pattern nearly impossible to identify by primary sequence alone. However with structural knowledge in hand as a guiding template, the 3D pattern becomes easily recognizable within the primary sequence of many M types. We hypothesize that just as with C4BP, hidden within the variable regions of M and M-like proteins are 3D patterns that are conserved for binding FH and Fg. We propose to test this emerging theme of conservation hidden within variability by unveiling potentially conserved FH- and Fg-binding 3D patterns in M and M-like proteins. We also propose to complete our work on C4BP, as the 3D pattern we discovered and published in Buffalo et al. explains only about a half of the set of M proteins implicated in C4BP binding. We propose to carry this out through X-ray crystallography. We have an extensive record of success with crystallographic studies of the M protein, and have obtained initial co-crystals of M proteins bound to C4BP, FH, and Fg. In each case, we will take advantage of the highly detailed level of information provided by our structural studies to determine the precise functional contributions of each of these interactions to virulence through our long-standing collaboration with co-Investigator Victor Nizet (UCSD). The results from our studies will provide essential guidance in designing anti-virulence strategies, and may have applications in the development of a broadly neutralizing GAS vaccine.